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Journal of Bacteriology, December 2005, p. 8520-8525, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8520-8525.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Coexpression Patterns of ^B Regulators in Bacillus subtilis Affect ^B Inducibility

Shuyu Zhang,^* Adam Reeves, Robyn L. Woodbury, and W. G. Haldenwang

Department of Microbiology & Immunology, MC7758, University of Texas Health Science Center, San Antonio, Texas 78229-3900

Received 5 July 2005/ Accepted 31 August 2005

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RsbT is an essential component of the pathway that activates the Bacillus subtilis ^B transcription factor in response to physical stress. rsbT is located within an operon that includes the genes for its principal negative regulator (RsbS) and the stress pathway component that it activates (RsbU), as immediate upstream and downstream neighbors. In the current work we demonstrate that RsbT's ability to function is strongly influenced by coexpression with these adjoining genes. When rsbT is expressed at a site displaced from rsbS and rsbU, RsbT accumulates but it is unable to activate ^B following stress. RsbT activity is restored if rsbT is cotranscribed at the alternative site with the genes that normally abut it. Additionally, an rsbS allele whose product allows constitutively high RsbT-dependent ^B activity displays this activity in rsbS merodiploid strains only when cotranscribed with rsbT and is recessive to a wild-type rsbS allele only if the wild-type rsbS gene is not cotranscribed with an rsbT gene of its own. The data suggest that RsbS and RsbT are synthesized in equivalent amounts and interact coincidently with their synthesis to form stable regulatory complexes that maintain RsbT in a state from which it can be stress activated.

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^B is a Bacillus subtilis sigma factor that directs RNA polymerase to promoters of the bacterium's general stress regulon (6, 8, 15, 23, 24, 28). General stress regulon induction occurs by the activation of ^B following exposure to physical or nutritional stress. In the absence of stress, ^B is held inactive in a complex with an anti-^B factor, RsbW (5, 7). ^B is released when a second protein (RsbV) binds to RsbW in lieu of ^B (5, 12). Although RsbV is normally present in the cell, it is unable to catalyze the release of ^B due to its inactivation by RsbW-dependent phosphorylation (12). Exposure to stress triggers one of two stress-responsive phosphatases to dephosphorylate phosphorylated RsbV (RsbV-P), reactivating RsbV to initiate the release of ^B from RsbW. One of the phosphatases (RsbP) responds to nutritional stress (glucose or PO₄ starvation, azide treatment), while the second (RsbU) is activated by physical stress (heat shock, osmotic shock, ethanol) (11, 17, 27, 28, 29, 30, 32, 35).

The physical stress phosphatase (RsbU) requires an additional protein (RsbT) for activity (35). In unstressed B. subtilis, RsbT is thought to be bound to an inhibitory protein (RsbS) in a high-molecular-mass complex (>10⁶ Da) formed by members of a family of homologous proteins (RsbRA, -RB, -RC, and -RD) (10, 20, 21). The RsbR complex appears to modulate the interactions between RsbS and RsbT (1, 2, 10, 14). In the absence of the RsbR proteins, RsbS is unable to inhibit RsbT's activation of RsbU (2). The roles of particular RsbR proteins are not known. They seem to be at least partially interchangeable in that the loss of any one RsbR protein does not significantly affect the activity of ^B (2). Only when multiple members of the RsbR family are lost is the ability of RsbS to inhibit RsbT compromised. Physical stress is believed to trigger RsbT to phosphorylate both RsbR and RsbS (9, 20, 35). As a consequence of these phosphorylations, RsbT is freed to activate RsbU and ultimately ^B (18, 35). Inhibition of RsbT is reestablished by RsbX, an RsbR-P/RsbS-P phosphatase which dephosphorylates these proteins and again allows the sequestration of RsbT (9, 28, 32, 35). The mechanism by which exposure to physical stress initiates the RsbT-dependent phosphorylation of RsbR and RsbS is unknown.

In recent work, we found that the large RsbR complexes present in crude bacterial extracts include RsbS but not RsbT (21). Although not part of the RsbR complexes, RsbT was also in a high-molecular-weight form that was Triton X-100 sensitive and likely an RsbT aggregate. In earlier studies (A. Dufour,U. Voelker, and W. G. Haldenwang, unpublished), it was observed that RsbT, unlike the other Rsb proteins, spontaneously aggregated during purification from Escherichia coli. This tendency to aggregate suggests that RsbT might be unusually sensitive to misfolding.

Although RsbS is viewed as a negative regulator of RsbT, it had been noted that an rsbS variant [rsbS(S59D)] whose product mimics the phosphorylated form of RsbS allows for higher RsbT-dependent ^B activity than does the loss of RsbS (17). This observation implied that RsbS may have an additional role in controlling RsbT's activity beyond merely sequestering it into an inhibitory complex. rsbT is located within the sigB operon, immediately downstream of rsbS (i.e., P_A rsbR rsbS rsbT rsbU, P_B rsbV rsbW sigB rsbX) (16, 33). The operon is expressed from a promoter (P_A) that is likely recognized by the bacterium's housekeeping RNA polymerase (E^A), with an internal ^B-recognized promoter (P_B) up regulating the downstream half of the operon during periods of ^B activity. Given the organization of the sigB operon and the tendency for RsbT to aggregate, a plausible secondary role for RsbS might involve facilitating the folding of RsbT or stabilizing it in the proper configuration for activity by a direct protein-protein interaction.

RsbT activity is influenced by coexpression with RsbR, -S, and -U. To explore the possible effect of the coexpression of rsbS with rsbT on RsbT's activity, plasmids carrying the sigB operon's P_A promoter joined to either rsbT alone, rsbR rsbS rsbT, or rsbR rsbS rsbT rsbU were placed at a site (spoIIG) within the B. subtilis chromosome separate from the sigB operon. All of the plasmids were constructed using a vector (pUS19) that contained an antibiotic resistance cassette (Spc^r) selectable in B. subtilis (4). The rsbT-expressing plasmid (pRW22) had been made in an earlier study (34). The remaining plasmids were constructed by cutting fragments containing either P_A rsbR rsbS rsbT or P_A rsbR rsbS rsbT rsbU from pRU13 (26) and cloning them into pUS19. To insert each plasmid into the B. subtilis chromosome at spoIIG, each plasmid was separately transformed (36) into a B. subtilis strain in which a plasmid (pUK19-1.1) (34) homologous with pUS19 had been previously inserted at the spoIIG locus. This strain and all of the other B. subtilis strains used in this study are congenic derivatives of PY22 (3). The integrated plasmid sequence served as a target for a single-site (Campbell-like) recombination of the rsbT plasmids into the chromosome at spoIIG. Recombination of the rsb-bearing plasmids into the resident pUK19 sequence was authenticated in transformation assays in which the anticipated linkage (>90%) between the antibiotic resistances carried by each plasmid was verified. Chromosomal DNA from representative clones with plasmid insertions at spoIIG were transformed into recipient strains that contained a ^B-dependent reporter system (SPß ctc::lacZ). DNA from the P_A rsbT plasmid-containing strain was transformed into BSW54, an RsbT^– strain [rsb(T63Q)term] (26). DNAs from representative P_A rsbR rsbS rsbT and P_A rsbR rsbS rsbT rsbU isolates were transformed into XS352, an RsbS^– RsbT^– strain (rsbST) (26). As a result of these manipulations, strains whose sole source of RsbT comes from a gene expressed either by itself or with other rsb genes at a site removed from the sigB operon were constructed.

Wild-type B. subtilis and the three strains expressing rsbT at spoIIG were grown in LB (25) and exposed to a condition (4% ethanol) that induces the physical pathway of ^B activation (32). Unlike the wild-type strain (Fig. 1A), the strain expressing only rsbT from the secondary site failed to respond to ethanol induction (Fig. 1B). Ethanol activation of ^B did occur if rsbR and rsbS were coexpressed with rsbT (Fig. 1C); however, the level of ^B activity in the P_A rsbR rsbS rsbT-expressing strain was consistently half of that seen when rsbU was also present in the transcription unit (Fig. 1D). These results reveal that coexpression of rsbT with the Rsb proteins, with which it is known to interact (31), affects RsbT's ability to respond to stress induction. Given that the loss of rsbR from the sigB operon does not block the inducibility of ^B by physical stress (33), rsbS is likely to be the critical component that is provided when RsbT's activity is restored by coexpression with rsbR and rsbS.

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FIG. 1. Expression of ctc::lacZ in complemented RsbT– strains. B. subtilis strains BSA46 (wild type, ctc::lacZ) (A), BSZ117 (PA rsbR rsbS rsb(T63Q)term rsbU, PA rsbT::spoIIG ctc::lacZ) (B), BSZ101 (PA rsbR rsbST rsbU, PA rsbR rsbS rsbT::spoIIG ctc::lacZ) (C), and BSW41 (PA rsbR rsbST rsbU, PA rsbR rsbS rsbT rsbU::spoIIG ctc::lacZ) (D) were grown in LB and exposed to ethanol (4%) at time zero. Samples of ethanol-treated () and untreated () cultures were taken at the indicated times (minutes) and analyzed for B-dependent ß-galactosidase levels (Miller units) (19, 25).

An obvious mechanism by which coexpression of rsbT with its potential interactors could affect RsbT's activity would be if this coexpression increased the abundance of RsbT. This might occur if synthesis of the upstream rsbS gene product enhances RsbT synthesis by translational coupling or if the synthesis of RsbS or RsbU in close proximity to RsbT facilitates their binding to RsbT to inhibit its degradation. To determine whether RsbT protein levels are influenced by its coexpression with the proximal rsb genes, Western blot analyses were performed on extracts prepared from the P_A rsbT-, P_A rsbR rsbS rsbT-, and P_A rsbR rsbS rsbT rsbU-expressing strains. Monoclonal antibodies specific for RsbR and RsbT (13) were used as probes in this assay so as to allow the levels of RsbR to serve as a standard for comparison of the relative RsbT levels in each of the extracts. A typical result is illustrated in Fig. 2. RsbT was found to be present in similar levels in extracts from each strain, regardless of whether rsbT was expressed alone or with its potential interactors. The data argue that expression of rsbT with its interacting Rsb proteins influences RsbT's potential activity without significantly affecting the levels of RsbT protein that are present in the cell.

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FIG. 2. Western blot analyses of RsbT levels in complemented RsbT– strains. Cultures of B. subtilis strains 1-BSZ117 (PA rsbR rsbS rsb(T63Q)term rsbU, PA rsbT::spoIIG), 2-BSZ101 (PA rsbR rsbST rsbU, PA rsbR rsbS rsbT:: spoIIG), and 3-BSW41 (PA rsbR rsbST rsbU, PA rsbR rsbS rsbT rsbU::spoIIG) were harvested during exponential growth (optical density at 540 nm [OD540], 0.2). Crude cell extracts were prepared and analyzed by Western blot analysis as previously described, using anti-RsbR, and anti-RsbT monoclonal antibodies as probes (13). The positions of RsbR and RsbT proteins in the blots are indicated.

RsbS preferentially controls the RsbT protein with which it is coexpressed. RsbT appears to be stress activatable if rsbS is coexpressed as part of its transcription unit, but not if rsbS is expressed from another site on the bacterium's chromosome (Fig. 1B and C). This argues that RsbS and RsbT likely interact with each other during or shortly after their synthesis. Such an interaction could be transient, whereby RsbS facilitates the proper initial folding of RsbT, after which each complex dissociates and both proteins become randomized in complexes with other RsbS and RsbT proteins. Alternatively, the initial interactions might be long-lived, perhaps separating only following stress induction. To explore these possibilities, we took advantage of an rsbS allele whose product [RsbS(S59D)] mimics RsbS's phosphorylated form and allows high ^B activity in the absence of stress (17). Integrating plasmids bearing the P_A promoter and either rsbR rsbS rsbT or rsbR rsbS were constructed. The rsbS allele in these plasmids was either wild-type rsbS or that encoding RsbS(S59D). The plasmids were transformed into wild-type B. subtilis, where they were expected to undergo single-site recombination into their homologous sigB operon sequences. There is a large region of homologous DNA upstream of the site of the rsbS mutation. This favors the isolation of transformants in which the rsbS(S59D) allele inserts into the B. subtilis chromosome as part of the truncated sigB operon originally carried by the plasmid. The recipient cell's intact sigB operon lies downstream of the RsbS(S59D)-encoding operon and separated from it by integrated vector DNA.

When grown in LB and exposed to ethanol stress the P_A rsbR rsbS rsbT merodiploid strains had distinct ^B activities (Pctc::lacZ), depending upon which rsbS allele was present as the second copy (Fig. 3A). The merodiploid strain with two wild-type rsbS alleles behaved like its wild-type parent (Fig. 1A), with a low background level of ^B activity and stress inducibility. In contrast, the strain with the rsbS(S59D) allele, although still stress inducible for ^B activity, had high background levels of ^B activity during growth. This composite phenotype is consistent with each of the rsbS gene products controlling a portion of the RsbT population. Under such a circumstance, wild-type RsbS would sequester a portion of the RsbT for stress-induced release, while RsbS(S59D) would allow constitutive activation of a portion of the RsbT. Given that RsbT's activity depends on its cotranscription with rsbS, RsbS and RsbT are likely to interact, concomitant with or soon after their synthesis. This suggests that the codominant phenotype displayed by each of the rsbS alleles is a consequence of each rsbS allele's effect on the rsbT product with which it is cotranscribed. To examine this possibility, the experiment was repeated using merodiploid strains that carried second copies of the rsbS alleles within operons that included rsbR but not rsbT. Now separated from rsbT, the rsbS(S59D) allele was unable to activate ^B in the absence of stress (Fig. 3B). The ^B-dependent ß-galactosidase levels of both the rsbS and rsbS(S59D) merodiploid strains were equivalent during growth and following ethanol induction. Thus, each of the RsbS proteins primarily controls the activity of the RsbT protein with which it is cotranslated.

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FIG. 3. B-dependent ß-galactosidase in rsbS merodiploid strains. B. subtilis PA rsbR rsbS merodiploid strains were grown in LB until an OD540 of approximately 0.2 was reached, at which point the cultures were split (arrows), ethanol was added to half of each culture to a final concentration of 4%, and incubation of both the ethanol-treated (open symbols) and untreated (filled symbols) cultures continued. Samples were taken at the indicated times (minutes) and assayed for B-dependent (Pctc::lacZ) ß-galactosidase activity (Miller units). The strains are as follows: BAR22 PA rsbR rsbS(S59D) rsbT () and PA rsbR rsbS rsbT rsbU () and BAR21 PA rsbR rsbS rsbT () and PA rsbR rsbS rsbT rsbU () (A); BAR34 PA rsbR rsbS(S59D) () and PA rsbR rsbS rsbT rsbU () and BAR35 PA rsbR rsbS () and PA rsbR rsbS rsbT rsbU () (B); and BSZ174 PA rsbR rsbS(S59D) rsbT () and PA rsbR rsbS rsb(T63Q)term rsbU () and BAR22 PA rsbR rsbS(S59D) rsbT () and PA rsbR rsbS rsbT rsbU () (C).

The ability of RsbS(S59D) to spontaneously activate RsbT when coexpressed with rsbT in a merodiploid strain, but not the RsbT synthesized with wild-type rsbS in a separate operon, supports the notion that each RsbS interacts with the RsbT with which it is synthesized and that, at least in the case of the wild-type RsbS-RsbT interaction, this association appears to be long-lived. If RsbT can readily be exchanged among RsbR/RsbS complexes, one would expect that the merodiploid strain that expresses the rsbS(S59D) allele in trans to rsbT would still be able to activate RsbT and exhibit an elevated background level of ^B-dependent ß-galactosidase activity. Instead, the uninduced ß-galactosidase levels found in a strain expressing rsbS(S59D) and rsbT in separate operons are equivalent to those seen in a merodiploid strain containing only wild-type copies of rsbS.

Although it is plausible that a complex between wild-type RsbS and RsbT could be long-lived, the RsbS(S59D) protein is believed to bind poorly to RsbT (35). This raises the question of why wild-type RsbS is not able to capture and silence the RsbT that is released from RsbS(S59D) to activate RsbU. Two possibilities seem likely: (i) either the RsbT that is passed from RsbS(S59D) to RsbU can no longer be reacquired by RsbS or (ii) RsbS and RsbT are normally synthesized at equivalent levels and as a consequence wild-type RsbS is already bound to RsbT and unavailable to capture the RsbT that is released from RsbS(S59D). To distinguish between these possibilities, a plasmid bearing P_A rsbR rsbS(S59D) rsbT was transformed into a B. subtilis strain with a null mutation [rsb(T63Q)term] in the sigB operon (26). In such a strain, the wild-type rsbS product does not have an RsbT binding partner cotranslated with it. If the RsbT released from RsbS(S59D) can be reacquired by RsbS, this RsbS should now be available to bind and silence it. In contrast, if the RsbT released from RsbS(S59D) cannot be acquired by the wild-type RsbS protein, ^B activity should persist. Figure 3C illustrates the ^B activities in the strains carrying P_A rsbR rsbT and the rsbS(S59D) allele with either a wild-type ^B operon or one with the rsb(T63Q)term allele. In the absence of an RsbT binding partner, wild-type RsbS blocks the heightened ^B activity that occurs when the P_A rsbR rsbS(S59D) rsbT sequence is paired with an intact sigB operon. Apparently, wild-type RsbS can acquire and hold inactive the RsbT expressed from the RsbS(S59D)-encoding operon. In this case, wild-type RsbS need not be synthesized with the RsbT that it ultimately silences. This argues that the inability of the wild-type rsbS allele to display dominance over rsbS(S59D) in a merodiploid strain, where both alleles are coexpressed with rsbT, is likely to be a result of insufficient wild-type RsbS to capture the RsbT that is made from both operons. The ability of RsbS to capture and hold in a stress-activatable state the RsbT originating from the rsbS(S59D) rsbT operon (Fig. 3C) but not the RsbT arising from an operon expressing rsbT alone (Fig. 1B) reveals that wild-type RsbS need not be cotranslated with RsbT for it to properly regulate RsbT's activity as long as the RsbT had been previously rendered activatable by coexpression with another RsbS protein, in this case RsbS(S59D).

The activity of rsbT, dependent on coexpression with its regulators, can be bypassed by overexpression. Although the current work indicates that coexpression of rsbS with rsbT is important for RsbT's stress-induced activity, we and others have found ^B activation to occur coincident with the induced expression of rsbT alone from a multicopy plasmid (17, 34). This argues that, under some conditions, RsbT can be synthesized in a form able to activate RsbU, without the need for cotranslation with RsbS. Presumably, under this particular circumstance, high levels of RsbT are being made from the multicopy plasmid relative to that generated from a single copy gene in the chromosome, which allows enough of this product to assume or maintain an active configuration and activate RsbU. To determine if, in fact, the levels of RsbT that are made under these two conditions are markedly different, RsbT abundance was evaluated using Western blot analyses. B. subtilis containing a multicopy plasmid (pRW13) (34) that carries rsbT under the control of an IPTG (isopropyl-ß-D-thiogalactopyranoside)-inducible promoter (P_SPAC) was grown in LB and exposed to IPTG (1 mM). Samples were taken at the time of IPTG addition and after ^B activity had become evident (Fig. 4A). Each sample was analyzed by Western blot analysis using monoclonal antibodies specific for RsbR and RsbT (Fig. 4B). Serial dilutions of extracts from wild-type B. subtilis (Fig. 4B, lanes 1 to 3) were also prepared for comparison to the dilutions from the induced culture (Fig. 4B, lanes 5 to 9). RsbR levels should be the same in both strains, allowing reactivity to RsbR to serve as a standard for comparison between the extracts. Quantitation of the antibody-reactive bands by densitometry (Alpha imager 2000; Alpha Innotech Corp., San Leandro, CA) revealed that RsbT levels, relative to RsbR, are 10-fold higher in the induced culture than the RsbT level present during growth in the wild-type culture. This order of magnitude increase in RsbT apparently provides enough active RsbT for some of it to encounter RsbU and activate ^B.

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FIG. 4. Induction of PSPAC rsbT from a multicopy plasmid. A B. subtilis strain, BSZ124, with an rsbT null mutation at sigB [rsb(T63Q)term] and a multicopy plasmid bearing PSPAC rsbT (pRW13) was grown in LB and treated with 1 mM IPTG at an OD540 of 0.13 (T0). Portions of the culture were harvested at the indicated times (minutes) for B-dependent (Pctc::lacZ) ß-galactosidase levels (Miller units) (A) and at the time of IPTG addition (T0) and 60 min after addition (T1) for Western blot analysis (B). (B) Western blot of crude extracts from an exponential culture of wild-type (BSA46) B. subtilis (lanes 1 to 3) and BSZ124 that was uninduced (lane 4) and IPTG treated (lanes 5 to 9). Lanes 1, 4, and 5 represent extracts from similar amounts (OD540 by volume) of cells. Lanes 2 to 3 and 6 to 9 are serial (twofold) dilutions of the extracts analyzed in lanes 1 and 5, respectively. The Western blots were probed, as described for Fig. 2, with anti-RsbR and anti-RsbT monoclonal antibodies.

In the original paper describing the construction of the rsbS(S59D) allele, it was suggested that RsbS might have an additional role in controlling RsbT activity beyond merely silencing RsbT in an inhibitory complex (17). The data in the current work support such a notion. A plausible but speculative model could envision RsbT as a protein prone to misfolding that must assume a particular configuration in order to activate RsbU. The active configuration may not be long-lived, decaying spontaneously and present in sufficient quantity to activate ^B only when RsbT levels are high or the active configuration is stabilized by an interaction with RsbS or RsbU. Thus, the absence of RsbS in the rsbS strain would result not only in the loss of RsbS's negative regulation of RsbT but also in the loss of the ability to facilitate the proper configuration of RsbT. In such a strain, some RsbT activity is presumably maintained by a direct interaction between RsbT and RsbU. Although RsbS is likely to be the Rsb protein with which RsbT normally associates, it is probable that in its absence RsbU binds to RsbT to preserve RsbT's activity. Not only do the above-mentioned B. subtilis strains with null mutations in rsbS display high ^B activity in the absence of stress, this phenotype is also seen in an engineered sigB operon with an additional promoter placed between rsbS and rsbT (17, 37). In both of these cases, rsbT expression occurs without rsbS as part of the transcription unit but with coexpression of rsbT and rsbU. In contrast with the rsbS strain, the rsbS(S59D) strain lacks the negative component of RsbS but potentially retains its positive contribution to RsbT structure. As a consequence, the rsbS(S59D) allele might be expected to promote greater ^B activity than the rsbS null allele. The finding that an rsbS deletion, but not the rsbS(S59D) allele, is tolerated within the sigB operon is consistent with such a model. The RsbR/S complex could then be thought of as a bifunctional entity, serving both as an inhibitory complex for RsbT and a stress-responsive chaperone-like system maintaining RsbT in a proper configuration and releasing it following exposure to stress. Given that chaperone activation and protein denaturation are key regulatory elements for triggering gene expression by physical stress in other systems (22), it is possible that the putative tendency of RsbT to misfold and aggregate, coupled with the ability of other Rsb proteins to influence this misfolding, may be relevant to the mechanism by which physical stress alters the Rsb proteins to modulate ^B control.

 
We thank Janelle Scott for construction of several of the plasmids used in this work.

This study was supported by U.S. National Institutes of Health grant GM-48220.

 
* Corresponding author. Mailing address: Department of Microbiology & Immunology, MC7758, University of Texas Health Science Center, San Antonio, TX 78229-3900. Phone: (210) 567-3956. Fax: (210) 567-6612. E-mail: zhangs{at}UTHSCSA.EDU.

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Journal of Bacteriology, December 2005, p. 8520-8525, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8520-8525.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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